LATENCY, LYSOGENY and SYMBIOSIS

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LATENCY, LYSOGENY and SYMBIOSIS

• LIVING WITH THE HOST

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What is latency?

• a period of quiescence (restricted or no
  replication or symptoms) that follows acute
  infection (virus replication w/wo symptoms that
  resolve) and has potential for repeat bouts of
  reactivation.
• host defenses are not effective (reduced or
  limited)
• repression of productive cycle genes
  restricted gene expression
• virus genome maintained intact

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Lysogeny occurs only in bacteria

• Common to all prokaryotes
• Reactivate to lytic by UV
• All dsDNA viruses
• Maintained and reproduce with cell
• Integrated or cytoplasmic
• Lysogenic conversion - new host phenotype due to
  expressed genes
• Superinfection immunity
• Insertional mutagenesis
• Cell wall structure
• Exotoxins

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Latency vs Lysogeny

• What cells
• What factors required
• State of the viral genome
• What genes are transcribed
• What proteins are expressed
• What are the conditions for reactivation

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Phage Lambda

• Nutrient rich medium goes
• lytic (lots of hosts)
• Nutrient poor medium goes lysogenic
• Stages in lysogeny
• Establishment
• Maintenance
• Induction

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Lambda genome
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Lytic cycle

• Transcription from two promotors
• IE products are N protein and cro protein
• N is an antiterminator for readthrough on the
  left and right

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• Delayed early on right is needed for DNA
  replication (O, P)
• Q product can turn on late genes
• Still uses host RNA polymerase

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• Same events on Pl and Pr
• Early termination without N
• N binds to transcript (not DNA) with host factors
  that read through to later termination signal

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Nut - N utilization site

• Forms structure on nascent RNA
• Binds with host proteins and RNA-P

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• Late genes made from new promotor Pr in presence
  of Q

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Establishment of lysogeny

• Readthrough from Pr makes CII
• Readthrough from Pl makes CIII
• CIII stabilizes CII from host protease Hfl
• CII binds to Promotor for Repressor Establishment
  (PRE) and activates CI (repressor) gene
• CII also activates genes for integration

pInt
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CI binds to operators on left and right

• Or 1-3
• Or 1gtOr2gtOr3
• Or1-2 block Pr and activate PRM (promotor for
  repressor maintenance)
• Or3 blocks PRM
• Ol blocks Pl

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Decision depends on Right side

• If CRO reaches high concentration before CI it
  binds to Or3 and blocks PRM
• At high CRO blocks Pr

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Induction results from destruction of CI

• DNA damage stimulates SOS response
• RecA protease cleaves CI
• CRO beats CI

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What evidence suggests that HSV is a latent virus?

• Can elicit HSV outgrowth by culturing neurons
  with appropriate cells.
• In situ hybridization
• PCR
• Why is latency a good strategy?
• Long term survival and immune escape
• Makes for an opportunistic pathogen

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HSV Lytic cycle

• Five IE genes - use host enzymes and host and
  viral transcriptional activators
• All IE promoters contain a common cis-acting
  sequence (TAATGARAT) that reacts with VP16
  tegument protein
• VP16 must interact with two cellular proteins,
  Oct-1 and HCF, to efficiently induce IE promoter
  activity
• Cell stimulated towards apoptosis but virus stops
  events through several IE gene products

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HSV establishes latency in terminally
differentiated nondividing sensory neurons.

• Virus infects and replicates in epithelial
  tissue, enters the neuron, travels via neuronal
  flow to cell body (regional ganglion)
• May have some replication
• DNA is circular episome returns via neuron
  (against the flow) to epithelial surface.

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• Effective immune system resolves primary
  infection and enhances establishment of latency.
  Recurrences diminish with time (Booster affect?)
• Reactivation triggers probably lead to permissive
  conditions some host factor? or reduced immune
  response.
• Evidence that transmission occurs without
  symptoms is there true latency? Is there some
  reactivation at all times?

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Latency associated transcript - major latency
product

• 8 kb processed to stable 2 and 1.5 kb
• nontranslated RNA
• Promotor for LAT
• has neuron specific elements.
• is antisense to one of the immediate early
  proteins, but can still get latency with fragment
  that does not overlap. Need 5 348 bp
• Is neuron IE nonpermissive cell?
• ICP4 binds and prevents LAT transcript in lytic
  cycle

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• Latency is thought to be passive no viral
  products are needed to maintain.
• Reactivation may require transient transcription
  and at least a few virions produced.This means
  the signal changes the transcription factors that
  are present.
• Levels of virus DNA in neuron is same in LAT and
  LAT-
• Conclusion Lat needed for reactivation not
  establishment

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• Recent evidence that LAT infection leads to
  increased neurovirulence (death of neurons)
• Does LAT protect cell from death during latency
  establishment or recurrences thus increasing
  number of infected neurons and allowing viral
  reactivation?

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LAT protects neuron from apoptosis

• LAT give large number of TUNEL neurons at 7
  days
• TUNEL (terminal deoxynucleotidyl
  transferase-mediated deoxyuridine TP nick-end
  labeling) fluorescent/chromogenic label bind to
  ends of DNA so fragmented DNA gets more label.
• A and D uninfected
• B and E WT HSV
• C and F lat- HSV

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Protection from apoptosis inducers

• Infected three different types of cells with a
  B-gal gene expression plasmid and LAT (APALAT)
  or LAT- (BABE) in the presence and absence of
  apoptosis inducers.
• If LAT reduces apoptosis then more cells with
  inducer will live and thus there will be more
  bgal activity than in absence of LAT gene.
  Compared to control plasmid with baculovirus
  antiapoptosis gene(CplAP)

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• The three apoptosis inhibitors work at different
  points in the pathway
• Protein kinase
• TNF
• Topoisomerase
• Thus LAT blocks something further in the pathway
• Two other HSV genes are antiapoptotic in
  productive infections.

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Apoptosis - Programmed cell death

• the death receptor-mediated pathway (Fas or TNF
  receptor)
• DNA damage through mitochondrial pathway
• ability of LAT to interfere with apoptosis
  correlates with its ability to promote
  spontaneous reactivation
• LAT enhances neuronal survival because it has
  antiapoptotic activity

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The concept of viral symbiosis

• Parasitoid wasps - use insect hosts to develop
  their larvae
• PolyDNAviruses (PDVs) needed for success
• PDVs produced in wasp ovaries and injected into
  insect with eggs
• Viral gene expression in insect - products
  manipulate host immune defenses
• No viral replication in insect host
• Endogenous provirus integrated in wasp genome
  and lost ability to be independent
• May go back 70 million years ago - vertical
  transmission

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First PDV genome sequenced Oct 2004

• 570,000 bp
• Composed of 30 DNA circles
• 156 coding sequences (white)
• Only 27 genome is coding
• 42 of coding DNA has no known homology
• Some known genes
• Protein tyrosine phosphatases (signal
  transduction?)
• Transcription factor regulators
• Immunosuppressive proteins
• Some genes look like modified host genes

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So what makes this a virus?

• Particles in insect
• DNA may have been from derived from host
• PDV DNA in wasp DNA at different regions
• May have been able to encapsidate in viral
  protein
• May be a virus that transferred replicative
  information to wasp
• Lost unneeded functions